The present invention relates to a method and an apparatus for the production of glass tubing having a narrowed diameter, and more in particular, glass microcapillaries. Microcapillaries when filled with an electrolyte have utility as microelectrodes or as patch clamp electrodes useful in electrophysiology and in the study of cell biology. More particularly, the present invention relates to microcapillary tubes having ultrafine tips with relatively short tapers.
In the study of membrane phenomena, particularly of nerve and muscle cells, valuable information may be obtained by measuring the potential difference across the cell membrane. The potential is typically measured by a high-impedance voltmeter, one terminal of which is placed in contact with extracellular solution, while the other terminal is placed in contact with the intracellular fluid. In order to make connection with the intercellular fluid it is necessary to penetrate the cell membrane to insert the electrode. The penetration must be accomplished without causing serious injury to the cell, or disrupting the cell structure or function. Electrodes formed of finely drawn-out glass tubes having an outside diameter of one micron or less filled with an electrolyte, such as an aqueous solution of salts of sodium or potassium, have been found to be aptly suited to use. However, as noted below, such electrodes have hitherto been restricted in use because of tube diameter. Such electrodes are sometimes referred to as micropipettes or ultra microelectrodes as the size of the electrode tip is past the resolving power of an ordinary light microscope.
The study of cellular response, especially in the field of neurophysiology, has heretofore been confined to the study of large cells because of the difficulties in penentrating small cells and maintaining normal activity after entry. Such limitation is particularly severe in the vertebrate brain, spinal cord, and retina where the vast majority of cells are smaller than about 20 microns in diameter. Usually cells show no noticeable injury when they are impaled with a microelectrode having a tip diameter of 0.5 micron or less. Such ultrafine electrodes have heretofore been produced, but only with utmost difficulty.
Microcapillaries may be manually produced by heating a small diameter glass tube until the glass becomes plastic and quickly drawing the tubing apart. However, this process requires considerable skill and more than an equal amount of patience. Even with those criteria the product produced manually varies widely in size and shape and the percentage of usable product is extremely low.
Various apparatus have been suggested to pull heated, small diameter, or capillary glass tubing to produce miocrocapillaries. For example, the following journal articles describe a number of such approaches: Rev. of Sci. Int., Vol. 24, No. 7, 1953, pp. 528-531 and Vol. 39, No. 2, 1968, pp. 158-60; J. Sci. Inst., 2, 1969, pp. 1087-1090; Neuroscience, 2, 1977, pp. 813-827; and J. Neuro. Methods, 7, 1983, pp. 171-183. Although the apparatus described produce a more consistent product than manual production, the product produced typically has an undesired taper or an outside diameter too large for small cells, i.e. greater than 0.5 micron. The taper, or bevel of a microcapillary tube is important as not only does the tip of the tube penetrate the cell but necessarily a portion of the tube length also enters the cell. Thus, a microcapillary with a sharp taper defeats the purpose of the narrow outside diameter at the tip. Conversely, a prolonged taper yields a microcapillary tube too fragile for normal use. Useful tips generally taper, as measured from the tube tip to the untapered portion of the tube, at least about twice the diameter of the starting tube.
Although the present invention is particularly adapted to the consistent production of microcapillary tubes having fine tips, those having outside diameters in there range between about 0.1 and 0.5 microns, microcapillary tubes of other useful configurations and sizes may be produced. For example, microcapillary tubes having tips ranging in size from about 0.5 to 1.5 micron outside diameter may consistently be produced. Such tubes are useful in extracellular patch recording electrodes. The taper angle in such tubes is usually steep to lower the series resistance and to facilitate the release of air bubbles. the dielectric loss and hence the noise of the recording is in part related to the thickness of the tube wall. The present invention provides a means of lowering capacitance and loss by controlling the forming conditions to produce a thicker tube wall. Microcapillary tubes having larger tips, between about 5 and 30 microns are useful in extracellular stimulating techniques, for suction pipettes, for injecting DNA or RNA into oocytes and for local fluid perfusion pipettes. Such microcapillary tubes of consistent shape and size are aptly produced by the present invention.